The role of cytochrome P-450 in ergot alkaloid biosynthesis will be investigated. The time-course for biosynthesis of agroclavine hydroxylase activity, cytochrome P-450, and NADPH-cytochrome c(P-450) reductase activity, will be determined. The conversion of agroclavine to elymoclavine, the conversion of gamma, gamma-dimethylallyl-tryptophan to 4-(E-4-hydroxy-3-methyl-delta2-butenyl)-tryptophan (intermediate steps in ergot alkaloid biosynthesis), the 14 alpha-demethylation of lanosterol (in pathway of ergosterol biosynthesis), and N-demethylation of aminopyrine (reaction catalyzed by rat liver microsomes), and inhibition of these reactions by carbon monoxide will be tested with the microsomal fraction from Claviceps purpurea PRL 1980. Cytochrome P-450 and NADPH-cytochrome c (P-450) reductase will be purified. The effect of the above substrates on the absorbance spectrum and EPR spectrum of the purified cytochrome P-450 will be determined. The binding of elymoclavine, which is a feedback inhibitor of ergot alkaloid biosynthesis, will also be studied. The binding constants of the ligands will be determined from the absorbance difference spectrum. Monooxygenase activity with the above substrates in a reconstituted system of purified cytochrome P-450 and purified NADPH-cytochrome c (P-450) reductase will be determined. Relationships between the properties of the microsomal cytochrome P-450 monnoxygenase system of C. purpurea PRL 1980 and alkaloid production rates and relative concentrations of intermediates in cultures will be investigated.